At the time of diagnosis, most epithelial ovarian cancers (EOCs) are no longer dependent on single genetic determinants for growth and/or survival. Therefore, targeted therapies used as single agents are not likely to be effective in this disease. We hypothesized that focused second site lethality screens performed using a cogently designed siRNA library would help identify critical cooperating oncogenic pathways that could be targeted using combinations of novel biologies. We have used RNAi approaches to identify candidates that selectively enhance killing by EGFR-inhibitors, such as erlotinib and cetuximab, and by the Src-targeting agent, dasatinib (also known as BMS-354825 or Sprycel). We have used bioinformatics approaches to map the pattern of hits back to a network of interacting signaling proteins. This has already revealed suggestive clusters of very closely interacting proteins, implying we have identified key survival nodes controlling resistance to drug treatment. In two cases, we have been able to exploit this information to develop novel, synergizing combinations of targeted therapeutic agents. The overall objectives going forward are to take the genes obtained through our siRNA screens, continue to map the sensitization network for targeted therapeutics relevant to EOC, and to design meaningful combinations of siRNAs with drugs, or drugs with drugs, that can be rapidly translated to the clinic. The four Aims proposed will systematically develop our preliminary studies to identify productive targets of co- inhibition, with the ultimate goal of identifying new drug combinations that will greatly enhance the treatment of women with EOC. Hence, Aim 1 will complete the initial hit validation process, create a master plate of individual hits and use this master plate of siRNAs to evaluate efficacy of the siRNAs in multiple EOC cell lines. In Aim 2, we will explore the expression patterns of proteins and transcripts for genes identified through hits in patient samples, to assess their clinical relevance. In Aim 3 we will perform animal-based experiments to further test supersensitizing combinations of drugs and siRNAs. In Aim 4 we will use the combined results of this analysis both to nominate new targets for drug development and to initiate clinical trials using combined molecular targeted agents. We believe that this cutting-edge approach will yield a paradigm that can subsequently be applied for multiple therapeutic applications. RELEVANCE (See instructions): Future clinical studies will need to explore schedules and combinations of treatments that optimize therapeutic results of existing agents, but at the same time novel druggable targets must also be found. The studies proposed offer an unprecedented opportunity to employ a functional approach to identify critical drug response-modifying genes that can be therapeutically targeted to improve ovarian cancer treatment outcomes with contemporary agents such as dasatinib and cetuximab, with or without the front line chemotherapeutic agents, platinum and paclitaxel.